Each sample was weighed (lg) and suspended in Ringers solution (lOml). The sample suspension was serially diluted (lml) in Ringers Solution to 10-4 diluted of the original concentration. Thereafter of each dilution (lml) was transferred to sterile petri dishes.
The agar that was used for the pour plates was Ohio Agricultural Experimental Station Agar (OAESA). The agar was prepared (250ml) and autoclaved (20min). After autoclaving the agar was allowed to cool. Thereafter Streptomycin (Sigma) and Chlorophenicol (Merck) solutions (2ml) were added. The agar was then poured (20ml) into each petri dish, the plates were gently swirled and the agar was allowed to set. The fungal plates were then incubated for seven days at 28°C. After the incubation period the fungal plates were removed and the growth was noted. The fungal colonies were examined macroscopically and there features noted. The fungi were examined microscopically to identify the type of spores and mycelia present in the fungi.
A loop full fungal spore was suspended in a loop full of water and mounted on a sterile slide. The smear was allowed to air-dry which was followed by the addition of one drop
of Lacto phenol Blue. A cover slip was then gently placed on each slide. The slides were examined using at a 100x magnification.
Extraction of toxins
After weighing the samples acetonitrile (Merck)/4% potassium chloride solution (9:lv/v) (lOOml) was added. The samples were shaken for one hour in a Controlled Environment Incubator Shaker (New Brunswick Scientific, USA). Thereafter the samples were removed and transferred to a Buchner flask and filtered through a Whatman No.1 filter paper on a Buchner apparatus (model Al 17). The solid material was discarded and the filtrate transferred to a 250ml-separating funnel. The filtrate was defatted twice with iso-octane (50ml) (Merck) and gently inverted funnel. Two distinctive layers were formed, the bottom layer was retained in a 250ml conical flask and the top fat containing layer was discarded. The extract was transferred back into the separating funnel and freshly prepared saturated sodium bicarbonate solution (Merck)/distilled water (50ml) (3:2v/v) and Dichloromethane (DCM)(50ml)(Merck) was added. Inverted flask gently to avoid emulsion, after a few minutes two distinctive layers were formed: DCM layer (Neutral Fraction) and the aqueous layer (Acid Fraction).
The Neutral Fraction was extracted with three portions of DCM (25ml), filtering each portion through a bed of sodium sulphate anhydrous (Merck), (lOg) in a folded llcm filter paper in a small funnel placed in the neck of a rotary evaporator flask. The rotary evaporator flask was inserted onto a Buchi Rotary Evaporator (Model 6540/2PR, Swiss) speed adjusted at 4, with a water bath not exceeding 60°C. The residue obtained was dissolved with acetonitrile (2ml). A piece of previously soaked dialysis tubing (25mmx16mm) (Sigma)(25cm soaked in distilled water for one hour) was firmly tied on
one end to fonn a closed sac. The dissolved residue was transferred into the dialysis tube using a 2ml Pasteur pipette and a knot were tied at the top. The dialysis tube was placed in a boiler tube (15x25cm) containing acetone (Merck) (30%v/v) (50ml). The boiler tube was sealed and was placed on a shaking table overnight. After shaking the boiler tube was removed from the shaker. The dialysis tube was discarded and the dialysate was transferred into a 250ml-separating funnel. This was again extracted three times with DCM and then rotary evaporated the samples. The residue was redissolved with DCM and transferred into a 10ml screw cap vial.
The Acid Fraction was treated with 1M sulphuric acid (25ml) (Merck) solution, to neutralize the sodium bicarbonate and waited four hours until effervescence subsided.
Extracted three times with DCM, filtered through a bed of sodium anhydrous and rotary evaporated. Residue was redissolved with DCM (2ml), and stored in 10ml screw cap vial.
The vials containing the acid and neutral extracts was placed on a heated hot plate (50°C) and evaporated with a steam of dry nitrogen, after which it was used for the detection step.
THIN LAYER CHROMATOGRAPHY (TLC)-SEPARATION/DETECTION The Neutral and Acid extracts were resolubilised with of DCM(200111). Each aluminum backed TLC plate (10 x 10cm) (pre-coated with silica ge160G) (Merck) was faintly marked with a pencil to mark the origin. Thereafter each extract was spotted(20111) onto the origin of the plate in portions(51ll) and drying at each stage by gently blowing on to the spot. The first solvent system used was CEI. Placed the CEI (20ml) in a TLC tank (12x12cm) and equilibrated (30 min). Each plate was inserted into the tanks placing the
origin in the bottom left hand corner. The solvent was allowed to run 1cm before the top of the plate. Removed plate and marked the solvent run. The plates was allowed to dry, and was then inserted at right angels to the first run, into the second solvent TEF system in a different tank.
The plates were then sprayed by propping it against a large glass plate, standing in a vertical position in a fume cup hood. CTA (Sigma) was used for the Neutral plates and PRA for the acid plates. The spray gun was held (50cm) away from the plates and completely sprayed the surface of the plates with the spray reagents. Neutral plates were viewed after heating (2 min at 110°C) and Acid plates immediately after spraying. After spraying each plate the unknown metabolites was detected by its characteristic colored spots that was produced by the spray agents. Each spot was circled with a pencil and labeled according to the colors produced e.g. Purple (P). The concentration of each metabolite in the original sample was estimated by the intensity of the spot. The Retardation Factor(Ri)was calculated by using the following formula:
Distance of solute Rf= Distance of Solvent
The Rfvalues were calculated for the CEI and TEF runs, and were compared against standards that were visible with the spray agents used.
PREPARATIVE TLC - ISOLATION OF THE METABOLITES
Washed each glass plate (20x20cm) with distilled water, cleaned with acetone (100%) (Merck) and dried. Weighed out silica gel 60G (60g) (Merck, Germany) into a 250ml beaker and added distilled water (7OmI), mixed and added to Shandon Southern Unoplan Spreader (Shandon Southern Unoplan, USA). The slurry was than immediately
coated on the plates (0.3-mm thickness), which was than rested (lOmin), and baked (110°C) in an oven overnight.
After baking the plates, it was removed and allowed to cool. Thereafter carefully drew a faint straight pencil line, above the plate (l.5cm) to mark the origin. Each sample was redissolved in DCM (600IlI) and vortexed. The plates were then spotted with each sample (200IlI), in portions (101l1). Each concentrated spot had an interval of Icm.The TLC tanks (22 x22 cm) fitted with stainless steel lids were equilibrated (30 min) with TEF (lOOmI). The plates were then inserted plate and were left to run (2 hours). Plates were than removed and air-dried. A portion of each plate was sprayed with the detection agents mentioned in 2.4.4, by placing a glass plate on top of the silica plate leaving a portion exposed to the spray reagents (Fig 3.6). Detected the characteristic metabolites, which appeared as straight bands. The bands were than scored off the plates, omitting the sprayed portion. Each metabolite was eluted from the silica gel with DCM (500ml) in equal portions (5ml), and was filtered through a sintered funnel, placed on a rotary- evaporating flask. The eluent was than rotary evaporated. The residue was dissolved with DCM (2ml) and nitrogen dried. Each metabolite was respotted on a TLC plate (lOxlOcm) to confirm purification of the metabolite.